Vaccine for prophylaxis or treatment of an allergen-driven airway pathology

ABSTRACT

The present invention relates to a live attenuated Bordetella pertussis vaccine which is deficient for tracheal cytotoxin (TCT), pertussis toxin (PTX), and dermonecrotic toxin (DNT) for prophylaxis or treatment of an allergen-driven airway pathology.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 16/840,276 filed on Apr. 3, 2020 (now U.S. Pat. No.11,110,161, which is a continuation application of U.S. patentapplication Ser. No. 16/537,342 filed on Aug. 9, 2019 (now U.S. Pat. No.10,653,765), which is a continuation application of U.S. patentapplication Ser. No. 15/800,921 filed on Nov. 1, 2017 now U.S. Pat. No.10,420,827), which is a continuation application of U.S. patentapplication Ser. No. 14/609,461 filed on Jan. 30, 2015 (now U.S. Pat.No. 9,839,683), which is a continuation application of patentapplication Ser. No. 13/782,754 filed on Mar. 1, 2013 (now U.S. Pat. No.8,986,709), which is a divisional application of U.S. patent applicationSer. No. 13/266,561 (abandoned), which was filed on Mar. 30, 2012pursuant to 35 U.S.C. 371 as a U.S. National Phase application ofInternational Patent Application No. PCT/EP2010/055507, which was filedon Apr. 26, 2010, claiming the benefit of priority to European PatentApplication No. 09305371.8, which was filed on Apr. 28, 2009. The entiretext of the aforementioned applications is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a vaccine for prophylaxis or treatmentof an allergen-driven airway pathology.

BACKGROUND OF THE INVENTION

The pathogenesis of allergic asthma remains unclear, however, thecurrent understanding involves the expansion of CD4+Th2 cells, and abreakdown in tolerance to otherwise innocuous environmental allergens(Romagnani et al. J Allergy Clin Immunol 2004; 113(3):395-400). Geneticpredisposition, coupled with environmental influences appears to affectthe regular suppression of Th2-mediated responses. It has beenhypothesized that abnormalities in the maturation of the lung duringfetal and neonatal development may render the airways more susceptibleto environmental allergens, favoring polarization towards the Th2phenotype and thus, predisposing the individual to atopy and asthma.Allergen-driven production of IL-4, IL-5 and IL-13 are typical ofallergic pathologies and the secretion of such Th2-cytokines initiatesisotype class-switching of B cells towards IgE, increased mucusproduction and recruitment of eosinophils to the airways. Since CD4⁺Th2cells represent a coordinating ceil type in some allergies, it wassuggested that the induction of counterbalancing responses might preventthe subsequent development of atopic disease. According to thismodification of Strachan's hygiene hypothesis (Romagnani et al. Int ArchAllergy Immunol 1992; 98(4):279-85), microbial exposure may activateinnate immune pathways that alter Th1, Th2 and Treg responses. Thisresults in the suppression of T helper 2 cell expansion, and aconsequent inhibition of isotype switching to IgE. However, severalstudies have suggested that viral and bacterial infections play a rolein exacerbation of respiratory disease. For example, respiratorysyncytial virus and Th1 inducing virulent Bordetella pertussis infection(Ennis et al. Clin Exp Allergy 2004; 34(9): 1488-97) exacerbate allergicinflammation in animal models.

Gram-negative B. pertussis causes whooping cough, a severe respiratorydisease responsible for significant infant morbidity and mortalityworldwide. Although immunizations with either killed whole cell vaccines(Pw) or more recent acellular subunit vaccines (Pa) have had success, are-emergence of the disease in young adults has been reported (Das P.Lancet Infect Dis 2002; 2(6):322). Typically, B. pertussis does notacutely affect this age group; however, infected adults can act asreservoirs, and increase the likelihood of infants contracting thedisease prior to vaccination. Most current vaccination regimes requirethree doses, beginning at 2 months of age necessitating 6 months foroptimal protection. Therefore, there is a need for vaccines that inducestrong protection against B. pertussis in neonates.

Virulent B. pertussis infection exacerbates airway pathology in a murinemodel of allergen driven inflammation, despite the induction of Th1immunity (Ennis et al. Clin Exp Allergy 2004; 34(9):1488-97). Th2inducing Pa vaccines protect against B. pertussis-induced exacerbationof allergic asthma, but induce IL-13 both at a systemic and local level(Ennis et al. Clin Diagn Lab Immunol 2005; 12(3):409-17). In contrast,systemic immunization with Th1 inducing Pw inhibits allergic airwayresponsiveness (Mills et al. Dev Biol Stand. 1998; 95:31-41), suggestingthat protection from allergen-driven pathology is not simply modulationof Th1/Th2 responses, but is associated with the degree of airway damageat the time of priming, such that allergen priming via the respiratorytract airways during breakdown of the airway epithelial mesenchymal unitmay be a more significant factor than Th11/Th2/Treg polarization.Recently, a genetically-attenuated live vaccine against B. pertussis,BPZE1, has been developed as a candidate neonatal vaccine againstwhooping cough (Mielcarek et al. PLoS Pathog 2006; 2(7):e65). This liverecombinant B. pertussis strain induces strong local and systemic immuneresponses upon intranasal delivery. Administration via the nasal routemimics natural infection and is expected to promote long-lastingimmunity in children from 1 month of age (Mascart et al. J Immunology2003; 170(1):1504-9). Three virulence factors have been targeted forattenuation; pertussis toxin, tracheal cytotoxin and dermonecrotictoxin. Using allelic exchange, genes encoding these toxins were deletedor replaced with genetically inactivated analogues in order to induceprotection, without the severe pathology associated with wild-typeinfection. However, the influence of BPZE1 administration on third partyallergen priming and allergen induced pathology is not known.

SUMMARY OF THE INVENTION

The present invention relates to a live attenuated Bordetella pertussisvaccine which is deficient for tracheal cytotoxin (TCT), pertussis toxin(PTX), and dermonecrotic toxin (DNT) for prophylaxis or treatment of anallergen-driven airway pathology.

The present invention relates to a method for prophylaxis or treatmentof an allergen-driven airway pathology in a subject, comprisingadministering to said subject an effective amount of a live attenuatedBordetella pertussis vaccine, wherein said live attenuated Bordetellapertussis vaccine is deficient for tracheal cytotoxin (TCT), pertussistoxin (PTX), and dermonecrotic toxin (DNT).

The present invention also relates to the use of a live attenuatedBordetella pertussis vaccine, which is deficient for tracheal cytotoxin(TCT), pertussis toxin (PTX), and dermonecrotic toxin (DNT) in themanufacture of a medicament for prophylaxis or treatment of anallergen-driven airway pathology.

DETAILED DESCRIPTION OF THE INVENTION

Examples of allergen-driven airway pathology are allergic asthma, Hayfever, interstitial lung diseases including pulmonary fibrosis.

Interstitial lung diseases including pulmonary fibrosis, may be causedby occupational or environmental exposures. Without wishing to be boundby theory, a live attenuated Bordetella pertussis vaccine, which isdeficient for TCT, PTX, and DNT would reduce airway damage andremodelling during a period of environmental exposure (to the agenttriggering Interstitial lung diseases) and would also protect againstvirulent B. pertussis exacerbation of pulmonary fibrosis.

By “subject” it is meant a human. Typically the subject is a neonate, aninfant or an adult.

Live attenuated Bordetella pertussis vaccines which are deficient fortracheal cytotoxin (TCT), pertussis toxin (PTX), and dermonecrotic toxin(DNT) have been described in WO2007/104451 and in Mielcarek et al. (PLoSPathog 2006; 2(7):e6). Recent advances in the understanding of B.pertussis virulence at the molecular level have allowed to rationallydesign a strategy for attenuation by removing or altering genes that areinvolved in the pathogenesis of whooping cough. Three virulence factorswere genetically targeted: tracheal cytotoxin (TCT), pertussis toxin(PTX), and dermonecrotic toxin (DNT).

TCT is responsible for the destruction of ciliated cells in the tracheaof infected hosts and may thus be involved in the cough syndrome. TCT isa breakdown product of peptidoglycan in the cell wall of Gram-negativebacteria, which generally internalize it into the cytosol by the AmpGtransporter protein to be re-utilized during cell wall biosynthesis. B.pertussis AmpG is inefficient in the internalization of peptidoglycanbreakdown products. The B. pertussis ampG gene can be replaced by E.coli ampG. The resulting strain expressed less than 1% residual TCTactivity. Any heterologous ampG gene from gram-negative bacteria thatrelease very small amounts of peptidoglycan fragments into the medium,can be used in the present invention. Examples of suitable heterologousampG gene include, but are not limited to ampG gene from Escherichiacoli, Salmonella, Enteoabacteriaceae, Pseudomonas, Moroxelia,Helicobacter, Stenotrophomonas, Legionella.

PTX is a major virulence factor responsible for the systemic effects ofB. pertussis infections and is composed of an enzymatically activemoiety, called S1, and a moiety responsible for binding to target cellreceptors. It is also one of the major protective antigens. The naturalptx genes can be replaced by a mutated version coding for anenzymatically inactive toxin. This can be achieved by replacing Arg-9 byLys, and Glu-129 by Gly in S1, two key residues involved in substratebinding and catalysis, respectively. Allelic exchange can be used tofirst delete the ptx operon, and then to insert the mutated version. Thepresence of the relevant toxin in the B. pertussis culture supernatantscan be detected by immunoblot analysis.

Other mutations can also be made such as those described in U.S. Pat.No. 6,713,072, as well as any known or other mutations able to reducethe toxin activity to undetectable levels. Allelic exchange can also beused to remove the dnt gene. Although the role of DNT in the virulenceof B. pertussis is not certain, it has been identified as an importanttoxin in the closely related species Bordetella bronchiseptica anddisplays lethal activity upon injection of minute quantities.

In a preferred embodiment, the live attenuated Bordetella pertussisvaccine is the BPZE1 strain.

The BPZE1 strain has been deposited with the Collection Nationale deCultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du DocteurRoux, F-75724 Paris Codex 15, FRANCE) on Mar. 9, 2006 under the numberCNCM 1-3585.

Typically, live attenuated Bordetella pertussis vaccines of theinvention may also carry heterologous antigens. The live attenuatedBordetella pertussis vaccines may be used as vector, to bear at leastone further heterologous nucleic acid sequence encoding a protein ofinterest. Typically, the protein encoded by at least one furtherheterologous nucleic acid sequence is a protein for which the expressionis desired in the respiratory tract. Typically, the protein of interestmay be an antigen, such as a viral or a bacterial antigen, against whichan immune response is desired. Examples of live attenuated Bordetellapertussis vaccines carrying heterologous antigens have been disclosedfor example by Si Ying Ho et al. (Infection and Immunity, 2008, 76(1),111-119).

Formulation of the vaccines of the present invention can be accomplishedusing art recognized methods. The amount of vaccines of the invention tobe administered to a subject and the regime of administration can bedetermined in accordance with standard techniques well known to those ofordinary skill in the pharmaceutical and veterinary arts taking intoconsideration such factors as the adjuvant (if present), the age, sex,weight, species and condition of the particular subject and the route ofadministration. The administration of the vaccine is usually in a singledose. Alternatively, the administration of the vaccine of the inventionis made a first time (initial vaccination), followed by at least onerecall (subsequent administration), with the vaccine.

Typically the vaccines can be administered by nasal administration or byinhalation. This type of administration is low in costs and enables thecolonization by the live attenuated Bordetella pertussis vaccine of theinvention of the respiratory tract. Nasal administration may beaccomplished with a live attenuated Bordetella pertussis vaccine underthe form of liquid solution, suspension, emulsion. Solutions andsuspensions are administered as drops. Solutions can also beadministered as a fine mist from a nasal spray bottle or from a nasalinhaler. Gels are dispensed in small syringes containing the requireddosage for one application Inhalation may be accomplished with a liveattenuated Bordetella pertussis vaccine under the form of solutions,suspensions, and powders; these formulations are administered via anaerosol, droplets or a dry powder inhaler. The powders may beadministered with insufflators or puffers.

In the following, the invention will be illustrated by means of thefollowing example as well as the figures.

FIGURE LEGENDS

FIG. 1 . Attenuated B. pertussis BPZE1 reduces the severity of airwaypathology induced by sensitizing allergen. Representative morphologicalchanges at 38 days in bronchiolar transverse sections of lungs from (A)Non-sensitized, (B) OVA-sensitized, (C) OVA-sensitized and infected withB. pertussis, (D) OVA-sensitized and immunized with BPZE1. Airwayinflammation was detected using haematoxylin and eosin (H&E) staining offixed lung sections. Original magnification A, C, E & G×100. B, D, F&H×400.

FIG. 2 . Attenuated B. pertussis BPZE1 reduces the severity of mucushyperplasia to sensitizing allergen. Representative morphologicalchanges at 37 days in transverse sections of bronchioles from (A)non-sensitized, (B) OVA-sensitized, (C) OVA-sensitized and infected withB. pertussis, (D) OVA-sensitized and vaccinated with BPZE1. Airwayinflammation was detected using combined Discombes/Alcian blue/PASstaining on lung sections. Original magnification ×400.

FIG. 3 . Attenuated B. pertussis BPZE1 reduces the cell infiltrate ofBAL fluid. Effect of virulent BPSM infection, attenuated BPZE1 challengeand/or OVA sensitization on BAL composition 24 h after final OVAexposure. Negative controls were sham infected/sensitized with saline.BAL fluid was examined for the total cell number (A), or the presence ofneutrophils (B), eosinophils (C) or lymphocytes (D). Results areexpressed as mean ±S.E.M. of cell number. *P<0.05.

FIG. 4 . Attenuated B. pertussis BPZE1 reduces allergen-induced IgE.OVA-specific IgE in serum elicited in response to OVA sensitizationand/or challenge with virulent (BPSM) or attenuated (BPZE1) B.pertussis. Sera were collected on day 38 and OVA-specific serum IgElevels were measured by ELISA. Concentrations below 100 pg/ml wereconsidered negative. Results are expressed as mean antibodyconcentrations ±S.E.M. P<0.05.

FIG. 5 . Cell-mediated immune responses from splenocytes to OVA,elicited by OVA sensitization 10 days following prior exposure toattenuated (BPZE1) or virulent (BPSM) B. pertussis infection. Negativesymbols indicate sham sensitization or challenge with PBS. Cytokineresponses from similar cultures assayed are shown for (A) IL-5, (B)IL-10, (C) IL-13 and (D) IFN-γ. T-cell proliferation (E) represents Δcpmof spleen proliferation against OVA at 200 μg/ml following subtractionof background typically 2000-4000 cpm. Results are expressed as mean±S.E.M.

TABLE I Summary of pathological features of B. pertussis/allergensensitization. Tissue BALF Goblet cell Spleen cell culture Groupinflammation eosinophils metaplasia IL-5 IL-13 IL-10 IFN-γ OVA-IgEControl − − − − − − − − OVA ++ ++ ++ +++ +++ − − ++ ZeOVA + + + − + −++ + SmOVA ++ +++ +++ + − − − +++ BPZE1 − − − − − − − − BPSM − − − − − −− − Features of airway inflammation it non-sensitized (Control),OVA-sensitized (OVA), BPZE1-immunized sensitized mice (ZeOVA) orBPSM-infected sensitized mice (SmOVA).

EXAMPLE Abstract

This preclinical study examined whether the candidate B. pertussisvaccine BPZE1 influences third party allergen priming and pathology,using previously characterized animal models. Unlike virulent wildtypestrains, live attenuated BPZE1 did not exacerbate but protected againstallergen-driven pathology.

Abbreviations Used

OVA: Ovalbumin; BAL: Bronchoalveolar lavage; BPZE1: live attenuatedBordetella pertussis; Pa: Acellular pertussis vaccine; Pw: Whole-cellpertussis vaccine

Materials Immunization, Sensitization and Airway Delivery of OVA and B.pertussis

Eight- to twelve-week old, female BALB/c mice (Harlan, Oxon, UK) wereused and maintained according to the regulations and guidelines of theIrish Department of Health, and the Research Ethics Committee of theNational University of Ireland, Maynooth. Mice were exposed to livevirulent or attenuated bacteria, and sensitized to allergen duringinfection. Virulent B. pertussis BPSM or attenuated BPZE1 were culturedas previously described (Mills et al. Dev Biol Stand. 1998; 95:31-41).Attenuated or virulent strains at mid-log growth were administered tomice by aerosol. At the peak of infection (10 d) and at 24 d, mice weresensitized by intra-peritoneal injection of 100 μg/ml ovalbumin (OVA) inadjuvant (AlumInject™, Pierce, Ill.). Mice were challenged intra-nasallywith OVA (50 μg/ml) on days 24, 35, 36 and 37. Various control groupsreceived sham delivery of sterile PBS in place of the active agent(Ennis et al. Clin Exp Allergy 2004; 34(9): 1488-97).

Bronchoalveolar Lavage (BAL) and Respiratory Tract Histology

On 37 d, mice were sacrificed by lethal injection of sodiumpentobarbital and BAL fluid collected (Ennis et al. Clin Exp Allergy2004; 34(9):1488-97). Total leukocytes and differential cell counts wereperformed as described, using Diff Quik/Rapi-Diff II™ (TriangleBiomedical Sciences, NC, USA.). Lungs from non-lavaged mice were removedand fixed in 10% (v/v) formalin/PBS, embedded in paraffin, sectioned andstained with haemotoxylin/eosin (H&E), alcian blue (identification ofmucus), Discombes (identification of eosinophils), or periodicacid-Schiff (for assessment of basement membrane thickness).Histopathological changes were graded according to an establishedsemi-quantitative scoring system as mild, moderate or severe. Pathologywas scored by two independent observers without prior knowledge of thetreatment group as previously described (Ennis et al. Clin Diagn LabImmunol 2005; 12(3):409-17).

T Cell Proliferation Assay

Splenocytes from mice were prepared as previously described (Mahon etal. J Exp Med 1997; 186(11):1843-1851) and incubated for 72 h witheither medium (negative control), OVA (200 μg/ml), or concanavalin A (5μg/ml). Supernatants were removed at 48 h for cytokine analysis, andcultures received fresh medium. Cells were incubated for the final 6 hwith [³H]-thymidine and proliferation was measured by radioactivityincorporated by liquid scintillation.

Measurement of Cytokines and Antibody Responses

Analysis of IL-5, IL-10 IL-13 and IFN-γ from BAL fluid and splenocytesupernatant was carried out using Cytometric Bead Array Flex Sets (BDBiosciences, Franklin Lakes, N.J.) according to manufacturer'sinstructions, and analysed by flow cytometry (Becton-Dickinson, NewJersey, USA). Standard curves and raw data were generated for eachcytokine using FCAP Array v1.0.1 software (BD Biosciences). OVA-specificserum IgE was measured by ELISA as previously described (Morokata Tetal. Immunology 1999; 98(3):345-351) using a rat anti-mouse IgEmonoclonal antibody (BD Pharmingen, San Diego, Calif., USA). IgEconcentration was expressed as μg/ml after comparison to murine IgEstandards (BD, Pharmingen, San Diego, Calif., USA).

Statistical Analysis

Values for all measurements were expressed as the mean±standard error ofthe mean (SEM). Statistical analysis was performed using GraphPad Prism™software (GraphPad, San Diego, Calif.). Comparison was made using theKruskal Wallis test, or the Mann Whitney test as appropriate.Significance was denoted by P value<0.05.

Results The Attenuated B. pertussis BPZE1 Prevents ExacerbatedOVA-Driven Allergic Airway Pathology

Virulent B. pertussis can exacerbate third party allergen priming inanimal models (Ennis et al. Clin Exp Allergy 2004; 34(9):1488-97) andhas been associated with exacerbation of allergy in humans (Harju et al.Thorax 2006; 61(7):579-584). To assess the influence of attenuated B.pertussis on third party allergen priming, mice were primed withvirulent or attenuated strains of B. pertussis, and sensitized to OVA atthe peak of bacterial carriage (a model previously shown to uncover theinfluence of infection on allergen-driven inflammation). In the absenceof infection, OVA sensitized mice exhibited typical peribronchial andperivascular inflammation at day 38, which was not observed in naïvecontrol mice (FIGS. 1A and B). At this time point, pathology due tovirulent bacterial infection alone has resolved. Priming at the peak ofvirulent B. pertussis infection enhanced airway pathology when comparedto OVA sensitization alone, with mice displaying epithelial hyperplasiaand moderate mucus metaplasia (FIG. 1C). In contrast, minimal pathologywas observed in sensitized mice infected with attenuated BPZE1, comparedto those sensitized to OVA alone (FIG. 1D). An examination ofmucus-containing goblet cells demonstrated that prior immunization withBPZE1 in OVA-sensitized mice reduced mucus secretion and hyperplasia,compared to those sensitized to OVA alone (FIG. 2 ). Thus unlikeinfection with virulent B. pertussis, immunization with the candidatelive attenuated B. pertussis vaccine BPZE1 did not enhance, but reducedthe pathology associated with allergen sensitization.

The Attenuated B. pertussis Vaccine Strain BPZE1 Prevents OVA-DrivenAllergic Airway Inflammation

Immunization with live attenuated B. pertussis BPZE1 moderated thequality of the OVA-induced inflammatory influx to the respiratory tract.Control mice showed minimal cellularity in bronchoalveolar lavage (FIG.3 ), whereas OVA sensitization/challenge resulted in significantinfiltration by inflammatory cells (>3×10⁶ cells, FIG. 3A, p<0.05).There were few remarkable differences in the numbers of lymphocytes orneutrophils. BPZE1 immunization alone did not support a neutrophilinfiltration at 38 d and infiltration of neutrophils in combinedBPZE1/OVA sensitized mice was typically lower than either OVA alone orin combination with virulent bacteria, however this did not achievestatistical significance in this study. The key observations were thatprior infection with virulent B. pertussis increased cellularinfiltration compared to sensitization by OVA alone accompanied byincreased eosinophilia (FIG. 3C) as previously observed (Ennis et al.Clin Exp Allergy 2004; 34(9):1488-97). However in marked contrastimmunization with live attenuated BPZE1 prior to OVA sensitizationresulted in significantly reduced OVA driven eosinophil infiltration ofthe airways (FIG. 3C, p<0.05). Thus, the candidate live attenuated B.pertussis vaccine BPZE1 prevents OVA-driven allergic airwayeosinophilia—a key feature of inflammation in this model.

The Candidate Live B. pertussis Vaccine BPZE1 does not Enhance Serum IgEResponses to Sensitizing Allergen

OVA sensitization in mice is known to induce IgE and a powerful specificTh2 response, whereas B. pertussis infection induces a strong Th1response. However, pertussis toxin alone can elevate IgE concentrations.Therefore, it was important to explore whether attenuated BPZE1 had anadjuvant effect or enhanced allergen-specific IgE. The influence ofBPZE1 on allergic sensitization was examined by measuring theconcentration of OVA-specific IgE in serum from mice sensitized to OVA,infected with BPSM, immunized with BPZE1, or receiving combinations ofthese treatments (FIG. 4 ). OVA sensitization induced significant levelsof IgE as is well documented. Previously, a significant increase inOVA-specific IgE following infection with virulent B. pertussis W28 wasobserved in OVA-sensitized mice (Ennis et al. Clin Exp Allergy 2004;34(9):1488-97). IgE responses in mice exposed to attenuated BPZE1 priorto OVA sensitization did not differ significantly to those receiving OVAalone. However, in marked contrast, attenuated BPZE1 immunizationresulted in significantly reduced induction (p<0.05) of OVA-induced IgEcompared to mice infected with virulent BPSM in combination with OVAsensitization (FIG. 4 ). Therefore, the candidate live attenuated B.pertussis vaccine BPZE 1 delivered prior to allergen primingdemonstrated no enhanced IgE response as observed with B. pertussisW28¹¹ and BPSM.

The Live Attenuated B. pertussis Vaccine BPZE1 Modulates Recall CytokineResponses to Sensitizing Allergen

It is clear that attenuated BPZE 1 has a radically different effect onallergen-driven airway pathology compared to virulent B. pertussis. Inorder to uncover the mechanistic basis of this effect, the influence ofbacterial exposure on the pattern of allergen-induced immune responseswas characterized. Allergen-specific cytokine induction by spleen cellpreparations was assessed following immunization with BPZE1 and OVAsensitization/challenge in order to evaluate the influence of BPZE1 onallergen-induced priming. As expected, OVA sensitization alone inducedhigh levels of the Th2 cytokines IL-5 and IL-13 (FIG. 5 ) on recall toOVA. Neither virulent BPSM nor attenuated BPZE 1 alone induced anyrecall response to OVA (FIG. 5E), but did produce strong Th1 responsesto B. pertussis antigens. BPSM challenge did not modulate the immuneresponse to sensitizing allergen, with no significant reduction in OVAspecific IL-5, IL-13 or proliferative responses observed in miceco-sensitized to OVA, and no significant increase in IFN-γ (FIG. 5 ). Incontrast, attenuated BPZE1 altered the pattern of cytokines induced bysensitizing allergen. BPZE1 significantly reduced the levels ofOVA-induced IL-5 (p<0.005) and IL-13 (p<0.05), as well as OVA specificproliferative responses (p<0.001), but induced significantly increasedIFN-γ in response to OVA (p<0.05). In summary, BPZE1 did not promote Th2cytokine induction to third party antigen but rather modulated this to aTh1 like response.

Discussion

The present study used combined infection/sensitization models todemonstrate that an attenuated strain of B. pertussis, BPZE1, did notenhance but reduced allergen-driven airway pathology. Attenuated B.pertussis reduced allergen-driven lung eosinophilia and decreased theseverity of airway inflammation. Furthermore, BPZE1 prevented anincrease in OVA-induced IL-5 and IL-13 and modulated recall responses toallergen to a Th1 like response. BPZE1 demonstrated reducedallergen-induced serum IgE responses when compared to mice infected withvirulent B. pertussis prior to OVA sensitization (see Table I). Takentogether these data demonstrate that attenuated BPZE1 does notexacerbate allergen-induced airway pathology in a murine model andsupports the use of this candidate vaccine for populations where atopyis prevalent.

The hygiene hypothesis suggests that Th1-inducing infections may have aninhibitory effect on the development of atopy. However, previous studieshave demonstrated that virulent B. pertussis enhances the severity ofairway pathology (Ennis et al. Clin Exp Allergy 2004; 34(9):1488-97)despite induction of Th1 immunity. In contrast, systemic immunizationwith a Th1-inducing Pw vaccine inhibited allergic airway responsiveness,suggesting that protection from allergen-driven pathology is linked notjust to CD4⁺ T cell profile, but also to the degree of airway damage atthe time of priming.

The purpose of this study was to investigate whether immunization with agenetically attenuated strain of B. pertussis could protect againstOVA-induced airway inflammation. Previously, the potential of othervaccines to moderate the risk of atopy has been investigated and anumber of studies have found an inverse relationship betweenimmunization and an increased risk of allergic disease. Ennis et atfound that a Pw vaccine protected against B. pertussis-exacerbation ofOVA-induced airway hyperresponsiveness in a murine model of allergicairway inflammation (Mills et al. Dev Biol Stand. 1998; 95:31-41).Likewise, Grüber et at found no allergy-promoting effect in response tocommon childhood vaccines including pertussis vaccines (Grüber et al.Allergy 2008; 63(11):1464-72). The relationship between childhoodimmunization and the development of atopic diseases in apopulation-based sample of 718 adolescents found that live attenuatedvaccines inhibited the development of asthma and allergic diseases(Martignon et al. Pediatr Allergy Immunol. 2005; 16(3):193-200). Thecurrent study demonstrates that the vaccine candidate BPZE1 suppressesallergen-driven pathology through a mechanism that modulatescell-mediated responses against OVA at both a mucosal and systemiclevel.

IL-5-mediated recruitment of eosinophils to the lung contributes toallergen induced airway pathology by generating potent cytotoxicproducts, including major basic protein (MBP) and eosinophil peroxidase,which collectively contribute to tissue damage (Gleich G. J Allergy ClinImmunol 2000; 105(4):651-63). Infection with virulent B. pertussisexacerbates the extent of the OVA-induced inflammatory influx to therespiratory tract, with an increase in eosinophils (FIG. 3C),accompanied by a marked increase in the severity of airway pathology(FIG. 1G). Conversely, administration of attenuated BPZE1 prior toallergen sensitization resulted in a significant reduction in eosinophilinfiltration. This study demonstrates that BPZE1 prevents theadjuvant-associated increase in OVA-induced IL-5 (FIG. 1A) seen whenanimals are infected with virulent B. pertussis strains. IL-13 alsocontributes to the pathogenesis of asthma by promoting Th2 responses,increasing eosinophil recruitment, and contributing to IgE-mediatedinflammation (Humbert et al. J Allergy Clin Immunol. 1997; 99(5):657-65;Temann et al. Am J Respir Cell Mol Biol 1997; 16(4):471-8). AttenuatedBPZE1 significantly decreased OVA induced IL-13 in sensitized mice (FIG.5C). Airway mucus hypersecretion is also linked to IL-13 and is a majorpathophysiological feature of both allergic asthma, and whooping cough.It is not surprising therefore that mucus production mirrored IL-13levels in this study and was significantly reduced in sensitized micepreviously exposed to BPZE1 (FIG. 5C).

This study suggests that one, or a combination, of the attenuatedvirulence factors in BPZE1 (pertussis toxin, tracheal cytotoxin anddermonecrotic toxin) play a role in the adjuvant effect observed withvirulent B. pertussis strains, via the induction of either IL-5 orIL-13, or both. The protection against allergen-driven pathology seenhere is associated with three genetic modifications contained withinBPZE1 and modulation of the allergic immune response is consistent withsome versions of the hygiene hypothesis. However, the mechanismsunderlying the beneficial influence of attenuated BPZE1 onallergen-driven pathology may be multiple and inter-linked. Previousstudies have demonstrated a significant increase in total serum IgE as aresult of OVA sensitization in animal models (Holgate et al. J AllergyClin Immunol 2005; 115(3):459-465; Hamelmann et al. Allergy 1999;54(4):297-305). Here, allergen-specific IgE responses induced byrespiratory sensitization were significantly reduced in mice receivingattenuated compared to virulent B. pertussis. This is consistent withthe modulation of systemic immune responses to OVA induced by attenuatedBPZE1 away from IL-5 and IL-13 towards IFN-γ, a response which isassociated with reduced IgE (Lack et al. J Immunol 1994;152(5):2546-54). Nevertheless allergic airway inflammation is not simplya balance between Th1 and Th2 responses. Hansen et at have shown thatmodulation of airway CD4′ Th responses does not necessarily reduceairway pathology (Hansen et al. J Clin Invest 1999; 103(2):175-183. Itmight be that the key beneficial feature of BPZE1 is the combination ofa Th1 skewed response combined with the absence of induced airwaypathology. This is consistent with previous reports in whichexacerbation of airway pathology to allergen was associated withallergen printing during a period of airway damage or remodelling(Marsland et al. Clin Exp Allergy 2004; 34(8): 1299-306; Gern. J AllergyClin Immunol 2000; 105(2 Pt 2):S497-502). This combined benefit makeslive attenuated Bordetella pertussis vaccine which is deficient for TCT,PTX, and DNT an attractive candidate as a protective agent againstatopy.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

What is claimed is:
 1. A method for reducing allergen-driven mucusproduction in the airway of a subject with allergy, the methodcomprising the step of administering to the subject's respiratory tracta live attenuated Bordetella pertussis strain which colonizes andreplicates in the subject's respiratory tract without inducing symptomsof pertussis.
 2. The method of claim 1, wherein the live attenuatedBordetella pertussis strain is deficient in tracheal cytotoxin (TCT),pertussis toxin (PTX), and dermonecrotic toxin (DNT).
 3. The method ofclaim 2, wherein the strain comprises a mutated pertussis toxin (ptx)gene, a deleted or mutated dermonecrotic (dnt) gene, and a heterologousampG gene.
 4. The method of claim 3, wherein the heterologous ampG geneis an E. coli ampG gene that replaces the wild-type Bordetella strainampG gene.
 5. The method of claim 3, wherein the mutation of the ptxgene comprises a substitution of an amino acid involved in substratebinding and/or an amino acid involved in catalysis.
 6. The method ofclaim 5, wherein the substitution of the amino acid involved insubstrate binding comprises R9K and the substitution of the amino acidinvolved in catalysis comprises E129G.
 7. The method of claim 4, whereinthe heterologous ampG gene is the only heterologous gene in the strain.8. The method of claim 1, wherein the live attenuated Bordetellapertussis strain is BPZE1.